CMFDA is a cell-permeant fluorescent dye widely employed as a whole-cell-tracking reagent. Upon intracellular enzymatic conversion, it forms a stable, fluorescent adduct that allows long-term monitoring of cell proliferation, migration, and viability. CMFDA is particularly valuable in studies requiring sustained, non-toxic labeling of living cells for imaging and flow cytometry.

Usage Method (This protocol is for reference only):
1. Preparation of CMFDA Stock Solution
Remove the product from the refrigerator and allow it to equilibrate to room temperature in a dry environment before briefly centrifuging and opening the lid (the product is highly hygroscopic). Dissolve the CMFDA powder in anhydrous DMSO (DMSO must be of high quality, freshly anhydrous, to avoid affecting experimental results) to prepare a 10 mM stock solution. The stock solution concentration may be adjusted according to experimental needs. The stock solution is highly prone to decomposition upon exposure to water; it is recommended to aliquot, seal with parafilm, and store at -20°C in a dry environment, avoiding repeated freeze-thaw cycles.
2. Preparation of CMFDA Working Solution
Before use, dilute the stock solution with serum-free medium to a working concentration of 0.5–25 µM, and pre-warm the working solution to 37°C. The working solution should be freshly prepared and used immediately; it should not be frozen for storage.
Note: The working concentration may be adjusted according to different experimental purposes. Typically, for long-term staining (at least 3 days) or staining rapidly dividing cells, 5–25 μM dye is required. For short-term staining (e.g., cell viability assays), 0.5–5 μM is sufficient. Use the lowest effective concentration to avoid cytotoxicity.
3. Staining Methods
3.1 Suspension Cell Staining
1) Centrifuge to collect cells and remove the supernatant, then gently resuspend the cells in pre-warmed CMFDA working solution.
2) Incubate under normal cell growth conditions for 15–45 min.
3) Centrifuge and remove the CMFDA working solution.
4) Continue culturing the cells in fresh culture medium for 30 min.
5) Wash the cells. If fixation is required, refer to Step 4.
3.2 Adherent Cell Staining
1) Remove the culture medium.
2) Gently add pre-warmed CMFDA working solution.
3) Incubate under normal cell growth conditions for 15–45 min.
4) Replace with fresh culture medium and continue culturing for 30 min.
5) Wash the cells. If fixation is required, refer to Step 4.
4. Fixation and Permeabilization (Optional)
4.1 Before fixation, wash the cells thoroughly with PBS.
4.2 Fix the cells with 3.7% paraformaldehyde at room temperature for 15 min.
4.3 After fixation, rinse the cells with PBS.
4.4 If subsequent staining with other antibodies is required, the cells should be permeabilized. The fixed cells may be incubated in pre-chilled acetone for 10 min (optional).
5. Fluorescence Microscopy Detection
After washing the treated cells with PBS, observe under a fluorescence microscope. CMFDA exhibits green fluorescence, with Ex = 492 nm and Em = 517 nm.